Drynaria extractions for treating osteoporosis and their extraction process

ABSTRACT

Rhizoma Drynariae  extract (RDE) is used as therapeutical agent or in the preparation for Osteoporosis, which is characterized in containing over 30 percent of total flavonoids. Furthermore naringin contained in flavonoids is more than 30 percent and less than 100 percent. The RDE is applied for the therapy of osteoporosis or used for producing drugs treating osteoporosis. And methods of extracting the same are also related.

FIELD OF THE INVENTION

The present invention relates to Rhizoma Drynariae Extract (RDE), morespecifically to a RDE for the treatment of osteoporosis, and thepreparation process of such and the use thereof in the treatment ofosteoporosis. The present invention is also related to the method oftreating osteoporosis with the extract.

BACKGROUND OF THE INVENTION

Rhizoma Drynariae is the rhizome of Drynaria fortunei (Kunze) J. Sm orD. baronii (Chist) Diels, which is used for the treatment of bonefracture for a long time (Zhong Yao Da Ci Dian (Lexicon of TraditionalChinese Medicine), 1658–1660, Shanghai People's Press, ShangHai, 1979).Ma Ke-Chang et al., studied the influence of Rhizoma Drynariae on ratOsteoporosis model, the results showed that Rhizoma Drynariae extractpartically restrains the glucocorticoid-induced bone loss (Ma Ke-Changet al., Zhong Yi Zheng Gu (Bone Setting) 1992, 4, 3), but the activecomponent therein is unknown. Additionally, Zhou Tong-Shui et al.reported that the effective component of Rhizoma Drynariae for treatingbone injury is naringin and analogues thereof (Zhou Tong-Shui, et. al.,Zhong Cao Yao (Chinese Herbal Medicine) 1994, 25, 249). And they alsoperformed assaying in crude plant of Rhizoma Drynariae (Zhou Tong-shuiet al., Zhong Guo Yao Ke Da Xue Xue Bao (Journal of China PharmaceuticalUniversity), 1996, 27, (9), 540), but they didn't show the relationshipbetween the above-mentioned active components and the effect toOsteoporosis thereof.

Wu Ying-Pi et al. disclosed a method of extracting naringin from Citrusgrandis Osbeck var. tomentosa Hort, aiming to obtain the singlecomponent of naringin (Wu Ying-Pi et al., Zhong Cao Yao, 1988, 19, 452)only. Therefore, it is unknown whether or not such method can be fittedfor other medicinal materials.

SUMMARY OF THE INVENTION

The object of the present invention is to provide Rhizoma Drynariaeextract (herein after also referred as RDE), which is available for thetreatment of Osteoporosis.

The other object is to provide a composition comprising RDE and the useof which for the treatment of osteoporosis.

Another object is to provide a process of preparation of RDE.

One further object of the present invention is to provide a compositioncomprising naringin for the treatment of osteoporosis, and the treatmentmethod of osteoporosis with the composition.

The technical embodiments of the present invention are as following:

1. An extract from Rhizoma Drynariae, wherein the content of totalflavonoids, based on the weight of the extract, is 30% or more, and thecontent of naringin thereof, based on the weight of total flavonoids, is30% or more, and less than 100 percent.

2. The extract according to 1, wherein the content of total flavonoidsis 50% or more.

3. The extract according to 2, wherein the content of naringin is 90% orless.

4. A pharmaceutical composition comprises the extract of 1.

5. The composition according to 4, which is used for the treatment ofosteoporosis.

6. Use of the extract according to 1 in the preparation of medicamentsfor the treatment of osteoporosis.

7. Use of the extract according to 1 for the treatment of osteoporosis.

8. A preparation process of the extract according to 1, comprising:

-   -   1) extracting Rhizoma Drynariae with water, alcohol or a mixture        thereof;    -   2) adsorbing of the extractant with resin; and    -   3) elution of the resin which has absorbed the extractant with        water, alcohol or a mixture thereof.

9. The preparation process according to 8, wherein the extraction isconducted with water.

10. The preparation process according to 8, wherein the extraction isconducted with alcohol/water mixture.

11. The preparation process according to 8 or 10, wherein the content ofalcohol in the alcohol/water mixture is 40–90% (w/w).

12. The preparation process according to 8, 10 or 11, wherein thealcohol is selected from methanol and ethanol.

13. The preparation process according to 12, wherein the alcohol isethanol.

14. The preparation process according to 13, wherein the resin ismacropore adsorption resin.

15. The preparation process according to 8 or 14, wherein said resin iswashed by water after adsorption of the extractant, before the elutionwith alcohol or alcohol/water mixture.

16. The preparation process according to 15, wherein the extraction isconducted with alcohol/water mixture.

17. The preparation process according to 15 or 16, wherein the contentof alcohol in the alcohol/water mixture is 40–90% (w/w).

18. The preparation process according to 15, 16 or 17, wherein thealcohol is selected from methanol and ethanol.

19. The preparation process according to 18, wherein the alcohol isethanol.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows HPLC chromatograph of standard naringin as control.

FIG. 2 shows the UV spectrographs of standard naringin, the solution ofthe extract of Example 1 and blank solution.

FIG. 3 shows HPLC chromatograph of the extract of Example 1.

FIG. 4 shows the comparison of TLC chromatographs of the solutions ofthe extracts from different processes.

FIG. 5 shows HPLC chromatograph of the extract of Example 3.

FIG. 6 shows HPLC chromatograph of the extract of Example 4.

DETAILED DESCRIPTION OF THE INVENTION

In the following descriptions of the present invention, percentagerepresents weight percentage, unless specifically identified.

In the present invention, Rhizoma Drynariae may be the rhizome or thewhole plant of the plants from the genus Drynaria (Bory) J. Sm,Polypodiaceae S. F. Gary, such as Drynaria fortunei (Kunze) J. Sm, D.baronii (Chist) Diels, D. sinica Diels, and D. delavayi Chirst, et al.,or Davallia mariesii Moore ex Bak from the genus Davallia Sm, and thelike. In the present invention, plants used as Rhizoma Drynariae may befrom one single species or a mixture of multiple species as describedabove, and is not limited.

The inventors have conducted an intensive investigation and found thatparticular extract from Rhizoma Drynariae or the single compoundnaringin have the activity of improving osteoporosis. Among the extractpossessing activity against osteoporosis, the content of totalflavonoids is always more than 30%, and content of naringin in theflavonoids is more than 30%. The compound naringin per se also has theactivity of improving osteoporosis, whereas the efficiency thereof islower than the active extracts described above.

In the present invention, the total flavonoids contained in RDE shouldbe 30% (wt %) or more, preferably 40% or more, more preferably 45% ormore, and most preferably 50% or more. Though there is no particularupper limit for the content of total flavonoids, which can be rangedfrom 30% to 100% in the extract, however, while considering the balanceof the choice of process and cost, as long as content of totalflavonoids is 50% or more, the object of this present invention can bewell achieved. If the content is upper than 90%, it does not only costmore, but extra procedure is also needed. Therefore the most preferabletotal flavonoids content ranged from 50% to 90%.

According to the present method, the RDE of present invention alsocomprises polyphenol compounds. Although there is no evidence forpolyphenol compounds against osteoporosis, but the extract of presentinvention may preferrably contain polyphenols, for polyphenols have theactivity of anti-oxidization, therefore the polyphenol compounds may behelpful for the present extract in the use of preperation ofpharmaceuticals. The content of polyphenols may vary depending ondifferent processes, but the contents are normally ranging from 10% to50%.

In the present invention, naringin's content should be 30% or more,preferably 40% or more, and more preferably 50% or more, with respect tototal flavonoids, but naringin shouldn't be the only flavonoid of totalflavonoids, preferably no more than 90%, and more preferably no morethan 80%.

We have extracted the flavonoids containing extract of Rhizoma Drynariae(as defined above). The results of experiments and clinical trials haveshown that the Rhizoma Drynariae extract produced by the process ofpresent invention has reliable effects on osteoporosis.

Although it is not proved by any known theories, but it is reasonablethat the activity of anti-osteoporosis of the present extract is notonly rooted in naringin, but also some other components which may havethe synergic effects with naringin. Under the limitations of presentinvention, such components co-present with naringin and show theactivity of anti-osteoporosis.

The producing process of Rhizoma Drynariae extract is discussed hereinafter.

A typical producing process may comprise the following steps: [Crudedrug pulverizing]→water/alcohol decoction→resin adsorption→(waterwashing)→alcohol/water elution—post procession

Among the process, means to pulverize Rhizoma Drynariae are commonprocesses to those skilled in the art and are not limited. If thegranularity of powder is too coarse, the active components will not besufficiently extracted; if the granularity is too fine, the separationof extracted solution after decoction may be difficult. Such can bereadily determined by the experience of those skilled in the art, andwill not be limited.

In order to extract effective component, soaking extraction by waterand/or alcohol may also be adopted. However, since the effectivecomponent may not be lixiviated completely, heat extraction ispreferred, the heating temperature may range from room temperature tothe boiling point of extracting solvent.

The temperature in heat extraction is preferably conducted at theboiling point of extracting solvent (reflux).

In case when the crude drug is extracted with water along, theextraction is conducted under 100° C. for 0.5–3 hrs and repeated for 2–4times, while the volume of water is 5–20 folds by weight of crude drugeach time. In case when extraction is conducted with alcohol or alcoholaqueous solution under reflux temperature, the crude drug is extractedfor 0.5–3hrs and repeated for 2–4 times, while the volume of solvent is5–20 folds by weight of crude drug each time. The alcohol may bemethanol or ethanol, and ethanol is preferred from the viewpoint ofsafety. When adopted, the concentration of ethanol is preferably in therange of 20–90%. And all the data mentioned above are for referenceonly. It can be modified based on the experience of person of the art,and it should be understood that such changes are not beyond the scopeof the present invention.

If desired, the extractant obtained may firstly be filtrated, thenadsorbed by resin, which may be macropore adsorption resin, such as D₁₀₁resin (manufactured by Nankai University Resin Factory, Tianjin), AB-8resin (manufactured by Tianjin Bone-gel Factory), WLD resin(manufactured by the Chinese Traditional Medicine Institute of SichuanProvince) and CAD-40 resin (manufactured by Huabei Pharmaceuticals),etc. The method of this present invention is not limited by the model ofresins. The detailed description of resin adsorption is described inCN1072089A, and referred herein with the present invention andincorporated as a part of it.

According to the research of the present inventors, WLD resin has thehighest adsorption capability among the resins mentioned above, and ismuch easier to be eluted. So use of WLD resin as adsorbent is preferred.The ratio of the amount of resin to crude drug may range from 0.5–2:1(w/w), preferably 0.5–1.5:1, most preferably about 1:1, which can bewell determined by those of the art. If the ratio is less than 0.5:1, itis likely to adsorb incompletely. If it is more than 1.5:1, the adsorbedflavonoid may not increase much more, and may cause economicaldisadvantages.

While employing adsorption resin, according to the common knowledge wellknown by those of the art, the resin should be pretreated via knownmethods before using, for example the resin might be pretreated asfollowing:

-   -   packing the resin in a column,    -   rinsing the column with ethanol-conc. HCl (1:1) until the        effluent fluid is clear upon dilution with a equivolume of        water, then    -   flushing the column with hot water (about 80° C.) ten-folds        volume of the resin column, after that,    -   rinsing the column with 2% sodium hydroxide -equivolume of the        resin column, and finally    -   rinsing with water until the effluent fluid become neutral.

The resin is then reverse-rinsed with water to loose, and then uploadthe solution to be eluted.

During absorption, the flow-rate should be controlled to ensure completeadsorption. Generally, at the time of industrial manufaction, while acolumn (φ 360 mm) packed with 100 kg of resin is employed, theadsorption may be ensured to be complete if the flow-rate be adjusted to10–20 L·min⁻¹. In such case, if the flow-rate is more than 20 L·min⁻¹,it is hard to adsorb completely, the yield may be lowered; if it is lessthan 10 L·min⁻¹, it is disadvantageous for increasing the productivity.

After adsorption, the resin may be eluted by alcohol such as ethanol ormethanol, ethanol is preferred from the viewpoint of healthy. When usingethanol, the concentration of which is preferably 50–95%, and the mostpreferred is 70% (may±about 5%) in consideration of the reutilization ofethanol.

While eluting, the ratio of the amount of alcohol to crude drug mayrange from 2 to 10. If the ratio is less than 2, the elution isincomplete, if it is more than 10, the eluted components will notincrease further and is economically disadvantageous. In view ofmanufacturing, 2–5 folds is preferred.

After elution, the eluent is collected and concentrated after removingof ethanol for reutilize. Finally, brown or umber powder is obtained viaspray-drying, lyophilizing or normal drying and pulverizing. The powdercan be refined or directly filled into the proper preparations, such ascapsules, pills, tablets, granules, solutions or injections.

Relative density of the residue is adjusted to 1.10–1.18 beforespray-drying, and is adjusted to 1.3–1.4 before vacuum drying. Afterdrying, the product may be pulverized to fine powder to facilitate thefurther processes. Spray-drying and vacuum drying are ordinarytechniques of drying and is not limited.

The Rhizoma Drynariae extract of present invention as described abovemay be prepared into any pharmaceutical preparations suitable forclinical uses, such as capsules, pills, tablets, granules, solutions orinjections upon ordinary preparation procedures. The dosage of each typeof preparation may vary depending on the composition of formulation,status of the patient and clinical conditions. General, in case when thecontent of total flavonoids in RDE is 50%, the oral administrationdosage may be 0.1–5.0 g/day in 1–4 portions per patient.

The Rhizoma Drynariae extract of present invention has outstandingactivity of improving osteoporosis. The following Examples are providedto illustrate the invention and do not limit it in any way.

EXAMPLES

The flavonoids in the RDE is qualitative characterized by potassiumborohydride (KBH₄) color-reaction. And the elution termination isdetermined by thin layer chromatography (TLC). And ultraviolet (UV)spectrometry is applied to quantitative analysis of total flavonoids.High-performance liquid chromatography (HPLC) is applied toquantitatively analyze naringin.

Potassium borohydride color reaction: KBH₄ (about 5 mg) is added into atest tube containing about 5 ml of sample solution and shaken up, intowhich several drops of hydrochloric acid is added, the appeared cherryred or fuchsia shows the existence of dihydroflavonone (sensitivity 55μg/ml). Otherwise it is negative if it doesn't appear cherry red orfuchsia.

TLC: Naringin standard in methanol to the concentration of 0.5 mg/ml isused as control solution. Based on the TLC method in accordance withPharmacopoeia Sinica (1995) appendix VI B, each of 4 μl of control andtest solutions are applied to one silica gel-G plate, after that elutedwith the upper layer of the mixture of benzene-ethyl acetate-formicacid-water (1:12:2.5:3). After elution, the plate is taken out andaired, sprayed by aluminum chloride (AlCl₃) solution and finallydetermined under UV light at 365 nm. The test sample shows afluorescence blot same color as that of the control in the correspondingsite of the plate.

UV Spectrometry Quantitative Analysis of Total Flavonoids:

This analysis is based on the UV spectrophotometric method inPharmacopoeia Sinica (1995) appendix VA.

-   -   Preparation of control solution: 10 mg of Hesperidin standard,        which is desiccated under 105° C. to constant weight, is        precisely weighed and dissolved in methanol (100 ml) in a        volumetric flask (100 ml). (Final concentration: 0.1 mg/ml)    -   Standard curve: Standard solutions of 0.50 ml, 1.00 ml, 2.00 ml,        4.00 ml, and 5.00 ml are precisely pipetted and diluted with        methanol to 25 ml of volume respectively. Methanol is used as        the blank control. UV detection is performed in accordance with        appendix VA), and absorbency is determined at 284±1 nm. The        calibration curve is plotted with the X-axis of concentration        and the Y-axis of absorbency.    -   Sample solution preparation: testing sample (0.25 g), which is        precisely weighed, is dissolved in methanol (50 ml) in conical        flask, ultrasonic extracted for 30 minutes, and transferred to a        volumetric flask (100 ml) after cooling. And the conical flask        is washed with methanol the rinsing liquid is also transferred        into the volumetric flask. And the solution is diluted by        methanol to 100 ml, and then filtrated. The subsequent filtrate        is collected, and a precise volume 1 ml of the filtrate is        applied to a chromatograph column (packed with 0.5 g of        polyamide as solid phase, which has passed 60 mesh of shieve).        Then the column is eluted by methanol with a flow rate of about        0.4 ml/min. About 25 ml of eluent is collected and diluted by        methanol to a volume of 100 ml.    -   Measurement: According to the UV spectrophotometric method in        Pharmacopoeia Sinica (1995) appendix VA, the absorbency of the        sample solution is detected, and then the total flavonoids        concentration is read from the standard curve.    -   Instruments and reagents: ultraviolet spectrometry, model UV-260        (Shimazu Inc., Japan), hesperidin standard (from the National        Institute for the Control of Pharmaceutical and Biological        Products)

HPLC Determination of Naringin

The test is based on the HPLC method of Pharmacopoeia Sinica (1995)appendix VI D.

-   -   Chromatographic conditions: C-18-alkylsilane-bonded-silica-gel        is used as solid phase, a mixture of methanol-(36% acetic        acid)-water (35:4:65) is used as mobile phase, detecting        wavelength at 283 nm. Number of theoretical plates is calculated        from the peak of naringin, and should be no less than 3000.    -   Preparation of control solution: Naringin standard, which is        desiccated under 105° C. to constant weight, is precisely        weighed and dissolved in methanol and diluted with methanol to        the final concentration 50 μg/ml.    -   Preparation of sample solution: the product (ca. 0.2 g) is        precisely weighted and put into 150 mL conical flask, 60 mL of        methanol is added therein. After 30 mins of ultrasonic        extraction, the solution is taken out and cooled to room        temperature. Afterwards the sample solution is transferred into        a 100 mL volume volumetric bottle. The conical flask is washed        with methanol and methanol is combined into the bottle. The        obtained solution is diluted to 100 ml by methanol, shaked to        uniform and filtrated. The subsequent filtrate is collected, 2        ml of the filtrate is precisely pipetted into a 25 ml volumic        bottle and diluted with methanol to the volume to obtain the        sample solution.    -   Measurement: Control solution (10 μl) and sample solution (10        μl) is respectively injected into the HPLC system for analysis.    -   Instruments and reagents: HPLC: LC-6A (Shimazu Inc., Japan); UV        detector: SPD-6AV; data processor (C-R4A), sensibility of data        recorder 0.16AUFS; Chromatograph Column: Shimpack C₁₈ column        (CLC-ODS 150×6.0 mm i.d., 5 μm); Protecting Column: YMG C₁₈        column (10×4.6 mmi.d., 10 μm); Supercentrifuge: TGL-16G        (Shanghai Medical Analytical Instruments Factory); Ultrasonic        cleaner: SB 3200 (Shanghai Bi'nengxin Ultrasonic Company, Ltd)

Standards of naringin are obtained from National Institute for theControl of Pharmaceutical and Biological Products. Under the abovespectrum conditions, it is calculated that naringin contained in thepresent standards is more than 98% by normalized method (see FIG. 1).Under the present experimental conditions, the peak of naringin isappeared at the retention time of about 15.26 min.

All the reagents and solvents used in the examples are analytically puregrade. Water is double-distilled.

Example 1

100 grams of Rhizoma Drynariae was pulverized and decocted in 1500 ml ofwater for 1 hour, the extract liquid was removed and additional 1000 mlof water was added and detocted again. The two detoction liquids werecombined and filtrated. The filtrate was applied to a pretreated column(φ 30 mm) packed with 120 g of WLD resin at a flow rate of 4 ml/min, andthe terminated point of effluent collection was determined by KBH₄ colorreaction, that is, the sudden appearance of dihydroflavonone showed thesaturation of adsorption in the effluent-resin. After adsorption, thecolumn was washed with 500 ml of water and washing liquid wasnot-recycled. Elution was conducted with 400 ml of 70% ethanol. Thestarting and end point of elution was determined by KBH₄ color test,eluent was collected and ethanol was recycled. Residual was evaporatedon a water bath to give thick paste, which was then dried in vacuo toafford 1.88 g of extract. Thus obtained RDE had a total flavonoidscontent of 55.08% detected by UV spectrometry, and a naringin content of36.6% (i.e. 66.4% of the total flavonoids) determined by HPLC.

FIG. 2 shows the UV spectrum of the solutions of RDE, naringin, andblank control (X-axis: wave length; Y-axis: absorbency; 1: naringinstandard; 2: present extract; 3: blank). From the UV spectrum, greatdifferentiation can be observed between RDE and naringin. RDE has anabsorption peak at around 200 nm, whereas naringin has only a shoulderpeak at that wavelength. And there is also difference in the maximumabsorption wavelength.

The high-performance liquid chromatogram of the extract is shown in FIG.3, in which there is at least one another unknown compound co-existingin the extract. (X-axis: Retention time; Y-axis: Integral intensity).

Example 2

In the same manner as that of Example 1 except that 70% of ethanol wasused for reflux and elution solvent. A 1.69 g of extract was obtained,in which the total flavonoids content was 53.12%, and naringin is 32.5%determined by HPLC (61.2% of the total flavonoids).

The comparison of TLC for several extracting solutions extracted bydifferent processes are shown in FIG. 4, in which from left to right arechromatograms of extractant and extract of example 1, extractant andextract of present example, and naringin. From the results can we seethat the extracts are substantially same no wonder extracted by water oralcohol.

Example 3

In the same manner as that of Example 1 except that AB-8 resin was usedinstead of WLD resin. A 0.95 g of extract was obtained, with the totalflavonoids content of 61.80%. FIG. 5 shows the HPLC of the extract ofExample 3.

Example 4

In the same manner as that of Example 1 except that D₁₀₁ resin was usedinstead of WLD resin. A 1.07 g of extract was obtained, with the totalflavonoids content of 57.35%. FIG. 6 shows the HPLC of the extract of inExample 4.

From FIGS. 3, 4 and 5 can we see that although different types of resinwas used as the adsorbent, the composition and content ratio offlavonoids in the extracts are generally the same.

Example 5

In the same manner as that of Example 1 except that 10 g of RhizomaDrynariae power was used each time, and 5 g, 10 g, 20 g and 25 g ofresins were used respectively. 0.16 g, 0.20 g, 0.27 g and 0.27 g of theextract were obtained accordingly, wherein the total flavonoids contentsare 53.88%, 52.29%, 42.65% and 41.88%, respectively. The yield of totalflavonoids were 86.2 mg, 104.6 mg, 115.2 mg and 113.1 mg, respectively,and the total flavonoid ratio are 0.86%, 1.05%, 1.15% and 1.13%,respectively. The above data shows that the total flavonoids extractedfrom the same amount of crude drug is almost not increasing when theweight ratio of crude drug to resin is more than 1:2, which means thatthe adsorption is complete when the resin is 2× the amount of the crudedrug. When weight ratio of crude drug to resin is 1:1, the totalflavonoids in adsorbate is relatively higher: the extract has an isamount of 90.8% of the full adsorption, with the total flavonoidscontent of 52.29%. Therefore, a ratio at about 1:1 of crude drug: resinis preferred.

Example 6

A 40 g of pulverized Rhizoma Drynariae was decocted with 1000 ml ofwater for 1 hour (slow fire), and the decoctate was filtrated anddivided into four portions averagely. Four glass columns (φ 20 mm) werepacked with pretreated WLD macropore adsorption resin (10 g). Everyportion of decoctates was applied to one column respectively atdifferent flow rates of 2 ml/min, 4 ml/min, 8 ml/min and 16 ml/min, andKBH₄ color reaction was applied to test whether there was flavonoidglycoside in the effluent. Results are shown in table 1.

TABLE 1 Flow rate (ml/min) Effects Flavonoid in effluent 2 Completelyadsorbed − 4 Completely adsorbed − 8 Incompletely adsorbed + 16Incompletely adsorbed +

It can be concluded that the control of flow rate ensures completeadsorption.

Example 7

A 300 g of raw pulverized Rhizoma Drynariae was treated in the samemanera as Example 1 to obtain the filtrate. And the filtrate was dividedto three portions averagely, each was applied to the column (φ 40 mm,packed with 100 g of WLD resin), then eluted with ethanol of differentconcentrations. The eluent was concentrated and dried, the totalflavonoids contents were measured via UV spectrometry. The results areshown in table 2.

TABLE 2 Ethanol concentration (%) (Group) 0% 50% (1) 70% (2) 95% (3)Total flavonoids 0 1.668 1.888 1.587 (g) 0 1.672 1.843 1.531 0 1.6791.871 1.574 {overscore (x)} ± SD 0 1.673 ± 0.006 1.876 ± 0.023 1.564 ±0.024

It can be concluded that the concentration of ethanol affects the effectof elution, in which the concentration of about 70% is preferred.

Example 8

Raw powder of Rhizoma Drynariae (500 g) was treated as the same mannerthat of Example 1 to obtain the filtrate, which then is applied to acolumn (weight ratio of crude drug to resin is 1:1). After adsorption,the resin was eluted with 70% ethanol in the volume of 1×, 2×, 3×, 5×,7× and 10× amount of crude drug (v/w), the eluents were collectedseparately, from which the ethanol was recycled. Then the totalflavonoids was measured separately. The results show that the elution iscomplete when elution by 10 folds of ethanol, which is shown in table 3.

TABLE 3 70% ethanol 1x 2x 3x 5x 7x 10x Sorbent (%) 0.47 8.76 1.99 0.470.44 0.22 Flavonoids 0.21 53.11 64.26 51.42 24.87 6.72 (%) Flavonoids0.987 4652.436 1278.774 241.674 109.428 14.784 (mg) % of total 0.01673.871 20.304 3.837 1.737 0.235 flavonoids

Results in table 3 show that the total flavonoids can be elutedcompletely, when eluted by enough quantity of ethanol (10 folds). Whenthe quantity of ethanol is 5 folds of RD, from which the totalflavonoids is amounted to 98% of the total flavonoids.

Preparation Example 1 Capsule

To 180 g powder of RDE obtained in the same manner that of Example 1, 70g of starch was added. The two components were mixed well and thenfilled into 1000 capsules. Each capsule contains 180 mg of effectivecomponent.

Preparation Example 2 Tablet

To 180 g powder of RDE obtained in the same manner that of Example, 70 gof starch was added. Then the mixture was kneaded with a small amount ofaqueous CMC-Na and then pelletized, dried according to the ordinarymethods, into which a small amount of magnesium stearate was added andmixed well to produce 1000 tablets. Each tablet contains 180 mg ofeffective component.

Preparation Example 3 Injectable Formulation

To 180 g powder of RDE obtained in the same manner that of Example, 800ml of injectable distilled water was added. The resultant mixture washeated to solve, then filtrated by microporous filter membranes, storedfor 24 hours at 4° C.–8° C., then filtrated again, adjusted pH to 6.5and isotonic with medicinal grade aqueous HCl (12N) and NaOH (12N),finally injectable distilled water was added upto 1000 ml, sterilized byheating, and filtrated, then divide into ampoules (5 ml per ampoule).Each ampoule contains 180 mg of effective component.

Test Example 1 The Clinical Test of RDE of Present Invention on PrimaryOsteoporosis.

The drugs with the following dosages and contents are used unlessadditional explanations.

Extracts in this present invention: capsules of preparation example 1,each capsule contains 0.18 g of effective component. Positive controlmedicine: α-D₃, (synthesized by TEVA PHARMACEUTICALS, Israel, preparedby Sino-US Kunming BARKER NORTON PHARMACEUTICAL CO., LTD), each capsulecontains 0.5 μg of effective component)

Diagnostic criteria: Comprehensive Diagnostic Grades for PrimaryOsteoporosis, according to the diagnostic criteria established bySpecialized Committee of Osteoporosis of Chinese Association ofGeriatrics. (cf. Gu-zhi-shu-song-zheng [M], p169–171, LIU Zhonghou eds.Chemical Industrial Press, Guangzhou, 1992)

According to the diagnostic criteria mentioned above, 70 cases ofosteoporosis are randomly divided into treatment group with RDE andcontrol group with α-D₃. RDE is administrated 180 mg, per os, tid, for 6months-2 period of treatment. And α-D₃ is administrated 0.5 μg, per os,bid, for 6 months too.

Bone density of lumbar (L₂–L₄) and top-femur (collum femoris, trigonalarea of femur, trochanteric of femur) is checked respectively byDouble-Energy X-ray Bone Density Meter (LUNAR DPX-L DEXA bonedensitometer,US). The unit is g/cm². And an Ultrasonic Bone Densitometer(model 2000-SYS-220, METRA Co., Israel) is employed to check the bonedensity of mid-portion of tibia.

The comparison of bone density pre- and after-treatment in two groups:

1. The results of bone density of lumber (L₂–L₄) pre- andafter-treatment are shown in table 4:

TABLE 4 Bone density of lumber (L₂–L₄) before- and after- treatment intwo groups ({overscore (X)} ± s, g/cm²) Before- after- 3 months after- 6months Group treatment treatment treatment Treatment group (17) 0.973 ±0.124 1.038 ± 0.234 1.101 ± 0.303 Control group (17) 0.958 ± 0.180 0.954± 0.179 0.958 ± 0.145

The bone density increased after 3 or 6 months treatment for treatmentgroup, but it is of no significant difference (P>0.05). There is noimprovement in control group. Also there is no significant differencebetween the two groups.

2. The results of bone density of collum femoris pre- andafter-treatment are shown in table 5:

TABLE 5 Bone density of collum femoris before- and after- treatment intwo Groups ({overscore (X)} ± s, g/cm²) after- 3 months after- 6 monthsGroup Before-treatment treatment treatment Treatment 0.736 ± 0.079 0.750 ± 0.079**  0.828 ± 0.124** group Control 0.748 ± 0.095 0.744 ±0.100 0.744 ± 0.093 group Compared with before-treatment, *P < 0.05, **P< 0.01

There is significant increase in bone density of collum femoris comparedbetween after- and before-treatment within treatment group (P<0.05,P<0.01). But it is of no significant difference within control group.

3. The results of bone density of trigonal area of femur pre- andafter-treatment are shown in table 6:

TABLE 6 Bone density of trigonal area of femur before- and after-treatment in two Groups ({overscore (X)} ± s, g/cm²) after- 3 monthsAfter- 6 months Group Before-treatment treatment treatment Treatment0.585 ± 0.104  0.600 ± 0.100*   0.661 ± 0.159**^(#) group Control 0.582± 0.125 0.591 ± 0.124 0.581 ± 0.134 group Compared withbefore-treatment, *P < 0.05, **P < 0.01; compared between control group,^(#)P < 0.05

There is significant increase in bone density of trigonal area of femurcompared between after- and before-treatment within treatment group(P<0.05, P<0.01). The bone density of trigonal area of femur increasedafter 3 months of treatment with no significant difference (P>0.05). Andalso there is difference between treatment and control groups after 6months treatment (P<0.05).

4. The results of bone density of trochanteric of femur pre- andafter-treatment are shown in table 7:

TABLE 7 Bone density of trochanteric of femur pre- and after- treatmentin two Groups ({overscore (X)} ± s, g/cm²) after- 3 months after- 6months Group Before-treatment treatment treatment Treatment 0.642 ±0.120 0.665 ± 0.125  0.752 ± 0.175** group Control 0.606 ± 0.109 0.616 ±0.099 0.616 ± 0.094 group Compared with before-treatment, **P < 0.01;compared between control group, **P < 0.01

There is significant difference between before- andafter-6-months-treatment in treatment group (P<0.01). And also there issignificant difference between treatment and control groups after 6months treatment (P<0.01).

5. The results of ultrasonic bone density of pre- and after-treatment intwo groups are shown in table 8:

TABLE 8 ultrasonic bone density in two groups ({overscore (X)} ± s,g/cm²) Before- after 3 months after 6 months Item Group n treatmenttreatment treatment Young Treatment 16 −1.875 ± 0.703    −1.643 ±0.766**  −1.288 ± 0.857** adult Control 15 −1.993 ± 0.872   −1.953 ±1.181   −0.593 ± 1.209   Age Treatment 16 −1.400 ± 0.700     −1.200 ±0.701**^(#)   −0.956 ± 0.682**^(#) matched Control 15 −1.433 ± 0.788  −1.386 ± 0.908   −0.987 ± 1.068   Speed Treatment 16 3769.42 ± 77.61  3793.00 ± 81.81**^(#)  3836.33 ± 99.03**^(#) sound control 15 3770.51 ±89.45  3761.77 ± 129.66* 3804.48 ± 138.89  Compared with pretreatment,*P < 0.05, **P < 0.01; compared with control group, ^(#)P < 0.05

The difference values between bone density (ultrasonic) of treatmentgroup and those of young adults and age matched increased significantly(P<0.01). And the speed sound increase significantly self-comparedwithin treatment group (P<0.01) and within control group (P<0.05).

Test Example 2 Experimental Study of RDE on Osteoporosis Models in Rats

Observe the effect of RDE in this invention on both Tretinoin-inducedand Ovariectomized (OVX) osteoporosis model in rats.

[Material and Methods]

1. Drugs

RDE was produced according to Example 1 (Per gram extract is equivalentto 66.67 g crude drugs). RDE was dissolved in distilled water to aconcentration of 0.47 mg/ml before administration. And administrationwas conducted via gastric tube. Tretinoin is manufactured by ChongqingHuabang Pharmaceuticals, Ltd. and was suspended to the concentration of1.5% with distilled water. Control drug α-D₃ was manufactured by TEVAPHARMACEUTICALS, Israel, which is solved in distilled water to theconcentration of 0.03 μg/ml before using and administrated orally viagastric tube.

2. Animals

Tretinoin-induced osteoporosis rats: sixty female W istar rats,16-week-old, mean weight 234.85±15.32 g. Ovariectomized rats: sixtyfemale Wistar rats, 12-week-old, mean weight 183.46±12.45 g. All theanimals are obtained from the Experimental Animal Center of ChineseMilitary Medical Academy.

3. Reagents and Instruments

Bone Biomechanics Rheometer was manufactured by Stevens Company, GreatBritain (model QTS-25). X ray Bone-density-meter was manufactured byNorland Company, USA (model XR-26). The others are all obtained frommarket.

4. Dose Selections and Groups:

-   (1) Tretinoin-induced group: Sixty rats are randomly allocated to    six groups (10 rats per group). Blank group: control animals    received PSS/d (physiological saline solution) alone. Model group:    Tretinoin is given orally 70 mg/kg/d; α-D₃ group: both tretinoin and    α-D₃ are given orally 70 mg/kg/d and 0.002 μg/kg/d respectively; RDE    low-dose group: both tretinoin and RDE are given orally 70 mg/kg/d    and 54 mg/kg/d respectively; RDE medium-dose group: tretinoin 70    mg/kg/d and RDE 108 mg/kg/d; RDE high-dose group: tretinoin 70    mg/kg/d and RDE 216 mg/kg/d.-   (2) Ovariectomized (OVX) rats group: Sixty rats are randomly    allocated to six groups (10 rats per group). Blank group is the    normal rats. All the other groups are removed amphi-ovaries. Model    group received PSS only; α-D₃ group: α-D₃ is administrated orally    0.002 μg/kg/d; And RDE is administrated orally in the dose of 54,    108, 216 mg/kg/day to OVX rats respectively, corresponding to RDE    groups of low-dose, medium-dose and high-dose.

6. Methods:

The dose administered to each animal is calculated from the individualbody weight recorded at the end of each week.

Tretinoin-induced osteoporosis groups: Blank group received PSS duringthe course of experiment. The other groups commenced treatment on thesame day, designated study Day 1, and once-daily tretinoin orally givenfor 14 days, after which they stopped administrating tretinoin. Bothblank and model groups are given PSS in Day 15, and α-D₃ group is givenα-D₃, RDE groups are given the corresponding dose of RDE orally for 14days. The final dose is given in the Day 28 before sacrificed.Investigation of Bone Density and Bone-biomechanics are performed in theDay 29.

OVX rats groups: Both blank and model groups are given the same volumeof distilled water orally, All the other groups commenced treatmentaccording to the way mentioned above for 12 weeks after which animalsare sacrificed and investigation of Bone Density and Bone-biomechanicsare performed.

7. Measurements

-   7.1. Bone density measurements are performed on No.2–4 lumbar spines    and right thighbone of each animal.-   7.2. Bone biomechanics measurements: three point bending tests of    left thighbone of each animal are performed; load-deformation curve    is recorded to measure the biomechanics index. Experimental    parameters: span 20 mm, loading velocity 10 mm/min. Bone structural    mechanics parameters: breaking load, elastic load, maximum radius,    elastic radius, maximum energy absorption; bone material mechanics    parameters: firstly calculated the Femoral Section Inertial Moment,    then computed the maximum strain, elastic strain, maximum stress,    elastic stress and elastic modulus according to the formulae.    [Results]

1. Bone Density of Rats in Each Group (Table 9 and 10)

TABLE 9 Bone density of Tretinoin-induced osteoporosis in rats Bonedensity (g/cm²) Group n thighbone Lumber spine Blank 10 0.146 ± 0.0080.147 ± 0.005 Model 10 0.124 ± 0.006 0.129 ± 0.00  α-D₃ 10 0.137 ± 0.0040.135 ± 0.005 RDE low-dose 10 0.140 ± 0.005 0.135 ± 0.005 RDEmedium-dose 10 0.140 ± 0.007 0.149 ± 0.007 RDE large-dose 10 0.152 ±0.006 0.149 ± 0.011

TABLE 10 Bone density of Ovariectomized (OVX) rats group Bone density(g/cm²) Group n thighbone Lumber spine Blank 10 0.14 ± 0.008 0.13 ±0.009 Model 10 0.11 ± 0.007 0.11 ± 0.035 α-D₃ 10 0.13 ± 0.009 0.13 ±0.009 RDE low-dose 10 0.12 ± 0.005 0.12 ± 0.005 RDE medium-dose 10 0.12± 0.005 0.12 ± 0.010 RDE large-dose 10 0.13 ± 0.007 0.13 ± 0.004

From Table 9 and 10 it is concluded that femoral bone density of RDEgroups and α-D₃ group increased obviously compared with model group. Andthere is significant differences in elevated degree of femoral bonedensity compared between RDE groups and α-D₃ group.

2. Bone Biomechanics Measurements of Rats in Each Group

-   2.1 The influence on bone structural mechanics indicators, see table    11 and Table 12.

TABLE 11 Bone structural mechanics indicators of Retinoic acid-inducedosteoporosis in rats Maximum load Maximum strain Maximum energyobservation Elastic strain Group n (N) (mm) (N-mm) Elastic load (N) (mm)Blank 10 146.60 ± 30.80 1.54 ± 0.36 787.95 ± 130.57 159.10 ± 88.80 1.65± 0.20 Model 10  66.10 ± 11.00 0.64 ± 0.10 405.88 ± 160.06  53.60 ±19.80 0.44 ± 0.18 α-D₃ 10 103.20 ± 3.40  0.98 ± 0.14 635.73 ± 114.46100.40 ± 7.00  1.01 ± 0.14 RDE low-dose 10 123.60 ± 6.09  1.04 ± 0.13564.38 ± 138.89 105.70 ± 4.30  1.06 ± 0.09 RDE medium-dose 10 135.30 ±12.00 1.33 ± 0.22 655.13 ± 131.46 110.00 ± 12.60 1.56 ± 0.19 RDElarge-dose 10 145.80 ± 11.00 1.65 ± 0.25 769.62 ± 128.43 120.40 ± 8.70 1.75 ± 0.16

TABLE 12 Bone structural mechanics indicators of Ovariectomized (OVX)rats Maximum strain Elastic strain Maximum energy Group N Maximum load(N) (mm) Elastic load (N) (N) observation (N-mm²) Blank 10 118.3 ± 12.401.11 ± 0.05 96.2 ± 6.00 0.93 ± 0.05 575.09 ± 138.50 Model 10  76.8 ±25.00 0.68 ± 0.01 62.5 ± 2.50 0.48 ± 0.07 425.25 ± 86.10  α-D₃ 10 102.1± 2.90  0.96 ± 0.10 88.6 ± 12.7 0.84 ± 0.06 520.48 ± 108.44 RDE low-dose10 97.0 ± 6.50 0.71 ± 0.10 72.1 ± 7.80 0.67 ± 0.09 498.55 ± 162.80 RDEmedium-dose 10 110.4 ± 10.20 0.92 ± 0.06 91.6 ± 6.70 0.85 ± 0.06 543.65± 100.78 RDE large-dose 10 134.3 ± 9.50  1.10 ± 0.11 99.4 ± 4.60 0.93 ±0.05 532.37 ± 110.83

From above table 11 and 12, the results showed that all the bonestructural mechanics indicators in RDE groups and α-D₃ group increasedsignificantly compared with model group. Also there is significantdifferences in the elevated degree of all the bone structural mechanicsindicators compared between RDE groups and α-D₃ group. And we can seesome dose-effect relationship in RDE groups.

-   2.2 The effect on bone material mechanics indicators, see table 13    and 14.

TABLE 13 Bone material mechanics indicators of Tretinoin-inducedosteoporosis in rats Maximum stress Elastic stress Rigidity coefficientGroup n (N/mm²) (N/mm²) Maximum strain Elastic strain (N/mm²) Blank 10309.3 ± 102.8 233.2 ± 48.5  0.04 ± 0.009 0.04 ± 0.013 41301 ± 1359 Model10 122.24 ± 54.8  70.9 ± 20.5 0.02 ± 0.004 0.01 ± 0.004 14436 ± 2648α-D₃ 10 167.2 ± 44.5  177.6 ± 51.3  0.03 ± 0.009 0.03 ± 0.01  30561 ±9194 RDE low-dose 10 101.8 ± 3.1  79.9 ± 21.1 0.04 ± 0.004 0.02 ± 0.00514958 ± 2347 RDE medium- 10 236.1 ± 59.2  217.5 ± 79.2  0.03 ± 0.0040.03 ± 0.011 24919 ± 2616 dose RDE large-dose 10 307.9 ± 7.9  333.9 ±27.1  0.04 ± 0.001 0.04 ± 0.007 36893 ± 3704

TABLE 14 Bone material mechanics indicators of Ovariectomized (OVX) ratsMaximum stress Elastic stress Rigidity Maximum Elastic Area of boneGroup N (N/mm²) (N/mm²) coefficient strain Elastic strain mould cortesBlank 10 254.2 ± 32.50 197.5 ± 22.40 44383 ± 3022 0.04 ± 0.003 0.04 ±0.003 9516 ± 267 6.23 ± 0.52 Model 10 123.4 ± 43.50 99.7 ± 8.60  34620 ±11208 0.03 ± 0.002 0.01 ± 0.002 4742 ± 748 3.53 ± 0.56 α-D₃ 10 221.5 ±46.50 147.2 ± 11.00 27508 ± 8073 0.03 ± 0.006 0.03 ± 0.010  7992 ± 11324.13 ± 0.99 RDE 10 138.3 ± 41.80 151.1 ± 12.50 33114 ± 4718 0.04 ± 0.0040.03 ± 0.006 7233 ± 872 4.02 ± 0.07 low-dose RDE medium- 10 140.0 ±40.60 137.7 ± 10.50 32729 ± 3307 0.05 ± 0.002 0.04 ± 0.002 7546 ± 6134.68 ± 0.44 dose RDE 10 249.9 ± 18.70 208.7 ± 17.80 44019 ± 1999 0.05 ±0.003  0.04 ± 0.0.04 8768 ± 543 6.15 ± 0.37 large-dose

From table 13 and 14, the results showed that RDE elevated the bonemaximum stress, elastic stress, maximum strain, elastic strain andrigidity factor of osteoporosis model rats. And there is significantdifference in those indicators compared between RDE group and α-D₃group.

From the results mentioned above, we can see that RDE elevate the bonedensity, enhance bearing capacity and anti-impact capacity to improvethe bone structural mechanics properties, and also has activity toimprove geometric configuration and inner material properties.

INDUSTRIAL PRACTICABILITY

RDE in the present invention has the activity to elevate the bonedensity of osteoporosis patient, to suppress the bone absorptiondistinctly, and of anti bone-loss. Thereafter the object of treatingosteoporosis can be achieved.

1. An extract from Rhizoma Drynariae, obtained by pulverizing theRhizoma, agitating and extracting the pulverized Rhizoma in a mixture ofwater and alcohol, the temperature of which is below 100° C., whereinthe alcohol in the mixture is between 20% and 90% of the water andalcohol mixture by volume, wherein the content of total flavonoids,based on the weight of the extract, is 30 percent or more, and thecontent of naringin thereof, based on the weight of total flavonoids, is30 percent or more, and less than 100 percent.
 2. The extract accordingto claim 1, wherein the content of total flavonoids is 50 percent ormore.
 3. The extract according to claim 2, wherein the content ofnaringin is 90 percent or less.
 4. A pharmaceutical compositioncomprises the extract of claim
 1. 5. The composition according to claim4, which is used for the treatment of Osteoporosis.
 6. A preparationprocess for a Rhizoma Drynariae extract wherein the content of totalflavonoids, based on the weight of the extract, is 30 percent or more,and the content of naringin thereof, based on the weight of totalflavonoids, is 30 percent or more, and less than 100 percent, theprocess comprising the steps of: 1) Extraction of Rhizoma Drynariae withwater, or a mixture of water and alcohol wherein the alcohol in themixture is between 20% and 90% of the water and alcohol mixture byvolume; 2) adsorption of the extractant with resin; and 3) elution ofthe resin which has absorbed the extractant with water, alcohol or amixture thereof.
 7. The preparation process according to claim 6,wherein the extraction is conducted with water.
 8. The preparationprocess according to claim 6, wherein the extraction is conducted withalcohol/water mixture.
 9. The preparation process according to claim 8,wherein the content of alcohol in the alcohol/water mixture is 40–90%(wt %).
 10. The preparation process according to claim 9, wherein thealcohol is selected from methanol and ethanol.
 11. The preparationprocess according to claim 10, wherein the alcohol is ethanol.
 12. Thepreparation process according to claim 6, wherein the resin is macroporeadsorption resin.
 13. The preparation process according to claim 6,wherein said resin is washed by water after adsorption of theextractant, before the elution with alcohol or alcohol/water mixture.14. The preparation process according to claim 13, wherein the elutionis conducted with alcohol/water mixture.
 15. The preparation processaccording to claim 14, wherein the content of alcohol is 40–90% (wt %)of the alcohol/water mixture.
 16. The preparation process according toclaim 14, wherein the alcohol is selected from methanol and ethanol. 17.The preparation process according to claim 16, wherein the alcohol isethanol.
 18. An extract of Rhizoma Drynariae, prepared by the processof: 1) extracting Rhizoma Drynariae using a mixture of water andalcohol, the alcohol concentration being between 20 and 90% by volume;2) adsorbing the extract with a resin; and 3) after the extract has beenadsorbed by said resin, eluting the resin with a mixture of water andalcohol.
 19. A method of extracting Rhizoma Drynariae, comprising thesteps of: 1) extracting Rhizoma Drynariae using a mixture of water andalcohol, the alcohol concentration being between 20 and 90% by volume;2) adsorbing the extract with a resin; and 3) after the extract has beenadsorbed by said resin, eluting the resin with a mixture of water andalcohol.
 20. The method of claim 19 wherein the resin is eluted using amixture of water and alcohol, said alcohol being less than 50% of themixture by volume.
 21. An extract from Rhizoma Drynariae, obtained bypulverizing the Rhizoma, agitating and extracting the pulverized Rhizomain water, the temperature of which is under 100° C., wherein the alcohoiin the mixture is between 20% and 90% of the water and alcohol mixtureby volume, wherein the content of total flavonoids, based on the weightof the extract, is 30 percent or more, and the content of naringinthereof, based on the weight of total flavonoids, is 30 percent or more,and less than 100 percent.